The present invention relates to a novel gene, NBS1, and its gene product, nibrin. In addition, it relates to methods for detecting mutations or polymorphisms of the gene that are associated with Nijmegen breakage syndrome in patients. Such mutations may be used to diagnose a predisposition to the development of certain pathological conditions in these patients.
Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder, belongs to the group of inherited human chromosomal instability syndromes, that includes Bloom""s syndrome, Fanconi""s anemia and ataxia-telangiectasia (AT). All of these disorders are characterized by spontaneous chromosomal instability, immunodeficiency and predisposition to cancer, but have distinct cytogenetic features and sensitivities to specific DNA damaging agents (for review see Digweed et al., 1993, Toxicol. Lett. 67:259-281). NBS shares a number of features in common with AT, most notably, a specific sensitivity to ionizing radiation (IR) and a predisposition to malignancies, particularly lymphoid cancers. Based on these features, and the results of earlier somatic cell complementation studies, NBS has long been classified as a variant of AT (for review see: Shiloh, 1997, Annu. Rev. Genet. 31:635-662). However, NBS has been shown to be genetically distinct from AT (Cerosaletti et al., 1998, Am. J. Hum. Genet. 63:125-134) and results from mutations in a novel mammalian gene.
Clinically, NBS is characterized by developmental defects, immunodeficiency, chromosomal instability, and an increased incidence of malignancies. Developmental defects typically observed in NBS patients include a severe and progressive microcephaly, growth retardation, mild to moderate mental retardation, and primary ovarian failure. Humoral and cellular immunodeficiency with recurrent respiratory infections is a consistent finding in NBS, but patients show intra- and interfamiliar variability. The immunologic characteristics of NBS encompass both developmental defects in tissues where lymphocytes develop and cellular defects in the responses of these cells to stimuli.
NBS patients have normal karyotypes, but chromosomal abnormalities are observed in cultured lymphocytes. The most frequently observed cytogenetic aberrations are rearrangements involving chromosomes 7 and 14 as observed in AT patients (van der Burgt et al., 1996, J. Med. Genet. 33:153-156). The distribution of the breakpoints on these chromosomes is non-random and preferentially cluster around immunoglobulin and T cell receptor loci where recombination events involving double strand breaks occur during lymphoid development. Malignancies, predominantly of lymphoid origin, occur in NBS with a high frequency and at atypically young ages. The most common tumors are B-cell lymphomas.
Cultured cells from NBS patients have impaired responses to ionizing radiation including an increased frequency of chromosomal aberrations, reduced survival in colony forming assays (Taalman et al., 1983, Mutat. Res. 112:23-32; Jaspers et al., 1988, Cell Genet. 49:259-263), radio-resistant DNA synthesis (RDS) (Jaspers et al., 1988, Am. J. Hum. Genet. 42:66-73; Wegner et al., 1988, Clin. Genet. 33:20-32; Chrzanowska et al., 1995, Am. J. Med. Genet. 57:462-471), a failure to activate cell cycle checkpoints (Seyschab et al., 1992, Eur. J. Pediat. 151:756-760; Jongmans et al., 1997, Mol. Cell Biol. 17:5016-5022), and a delayed up-regulation of p53 levels (Jongmans et al., 1997; Matsuura et al., 1998, Biochem. Biophys. Res. Commun. 242:602-607). All of these features are also observed in cells from patients with AT (Shiloh, 1997, Annu. Rev. Genet. 31:635-662).
In order to understand the complex relationship between NBS and AT, cell lines from patients with these disorders have been fused and assayed for various phenotypes involving response to ionizing radiation. Complementation for RDS has been reported in AT/NBS cell hybrids (Jaspers et al., 1988; Wegner et al., 1988). However, complementation of RDS has also been observed in hybrids resulting from fusions of cells from different AT patients suggesting the existence of multiple complementation groups for AT (Jaspers et al., 1988)xe2x80x94a finding that has not been borne out by mutational analysis of the AT gene (ATM) (Savitsky et al., 1995, Science 268:1749-1753; Concannon and Gatti, 1997, Hum. Mutat. 10:100-107). Microcell mediated transfer of a normal copy of chromosome 11 containing the AT gene did not complement the radiation sensitivity of NBS cells suggesting that the gene mutated in NBS was not ATM (Komatsu et al., 1996, Am. J. Hum. Genet. 58:885-888). Non-complementation was also observed in AT/NBS hybrids tested for another phenotype, radiation induced chromosomal aberrations. This latter finding has been interpreted as suggesting that the products of the AT and NBS genes, although distinct, may interact in a common biochemical pathway, or may be parts of a common protein complex (Stumm et al., 1997, Am. J. Hum. Genet. 60:1246-1251).
Molecular cloning of a gene associated with NBS would facilitate the analysis of the underlying defects in NBS. While genome-wide search for linkage in NBS families localized the gene to a 1 cM region on chromosome 8q21, the large physical size of this region, and the small number of available NBS families with informative recombination events, limited further progress towards gene identification.
The present invention relates to a novel gene, NBS1, and its encoded product, nibrin. In particular, the invention relates to polynucleotides encoding nibrin or fragments thereof, nibrin polypeptides, antibodies to nibrin polypeptides, methods for detecting nibrin in a cell, and methods for diagnosing NBS by detecting expression levels or mutations of NBS1 gene, as well as methods of treating NBS.
The invention is based, in part, or Applicants"" discovery that a novel polynucleotide isolated from a region of human chromosome 8q21 contained mutations in all NBS patients. The mutations included deletions and insertions that resulted in a frameshift as well as a point mutation. Specific mutations associated with the NBS plenotype include 657del5, 698del4, 835del4, 842insT, 1142delC, 976C greater than T, 681delT and 900del25. In addition, polymorphisms of the gene include 553 G/C, 1197 T/C, 2016 A/G, 102 G/A, IVS 5+9 T/C, IVS5+51delT, IVS9+18 C/T and IVS-7A/G.
It is an object of the invention to detect a mutation or polymorphism in NBS patients. A mutation includes a missense, nonsense and frameshift mutation.
It is another object of the invention of the invention to diagnose a predisposition to a pathological conditions such as cancer, microcephaly, mental retardation, and primary ovarian failure, based on detection of a mutation in the NBS1 gene disclosed herein.
It is also an object of the invention to treat NBS by replacing the mutated gene in a NBS patient.